PROJECT SUMMARY Studies of developmental regulation of globin gene expression have provided important mechanistic insight into normal mammalian gene control and abnormal gene expression in diseases. Epigenetic mechanisms are now recognized as central to globin gene regulation as well as dysregulation of genes in leukemia and other cancers. This project is aimed at elucidating the key protein-protein interactions among components of the MBD2-NuRD chromatin remodeling complex in the context of fetal ?-type globin gene silencing in adult human erythroid cells. The long term goal is to identify and validate targets for safe therapeutic activation of fetal hemoglobin expression in sickle cell anemia and ?-Thalassemia. This goal will be pursued through the following collaborative aims: 1) To functionally define the roles of specific regions of MBD2 and NuRD complex components in the ability of MBD2-NuRD to silence the fetal ?-globin gene in adult human erythroid cells and 2) to biophysically and structurally characterize interfaces critical for MBD-NuRD complex formation and stability. The experimental approach employs a real-time iterative feedback between genome editing of key protein interactions in adult erythroid cells in conjunction with structural studies of key protein-protein interactions of MBD2, GATAD2A and CHD4 NuRD components using both standard NMR, and crystallographic techniques as well as state-of-the-art paramagnetic relaxation enhancement (PRE) and bioluminescence resonance energy transfer (BRET). The erythroid genes that are directly activated by disruption of MBD2-NuRD and in turn relieve ?-globin gene silencing will be characterized by both RNA-seq and ChIP-seq assays and bioinformatics analyses as well as ChIP assay validation. These experiments will identify and validate proof of principle peptide and small molecule targets for future development of therapy of sickle cell anemia and ?-Thalessemia.